Biochemistry 2007, 46, 2865-2875
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Vault Nanocapsule Dissociation into Halves Triggered at Low pH† Lisa E. Goldsmith,‡,§ Marcella Yu,‡,§ Leonard H. Rome,|,⊥ and Harold G. Monbouquette*,‡,⊥ Chemical and Biomolecular Engineering Department, Department of Biological Chemistry, and California NanoSystems Institute, UniVersity of California, Los Angeles, California 90095 ReceiVed March 29, 2006; ReVised Manuscript ReceiVed December 16, 2006
ABSTRACT: Vaults are self-assembled ribonucleoprotein nanocapsules that consist of multiple copies of three proteins (major vault protein, VPARP, and TEP1) and an untranslated RNA. Although their function has not been determined, vaults are found in nearly all eukaryotic cells. This study describes the use of fluorescence spectroscopy, multiangle laser light scattering (MALLS), and the quartz crystal microbalance (QCM) as tools in investigating recombinant vault conformational change in response to a varied solution pH. Identification of conditions for reversible vault disassembly and reassembly could enable application of these nanocapsules in drug delivery and in nanomaterials synthesis. Initial monitoring of changes in the intrinsic fluorescence intensity of vaults showed a 60% increase at pH 3.4 compared to that at pH 6.5, suggesting vaults exhibit a more open conformation at low pH. Fluorescence quenching studies provided further evidence of a vault structural change at low pH. MALLS data suggested a decrease in molecular mass accompanied by a clear increase in the radius of gyration as the solution pH was shifted from 6.5 to 3.4. This result prompted the hypothesis that vaults dissociate at least partially at low pH. Using the QCM to study adsorption of the vault onto self-assembled monolayers, data that suggest vault dissociation at low pH, even when the vault is in an adsorbed state, were also obtained. Finally, transmission electron microscopy (TEM) of negatively stained vaults at pH 6.5 and 3.4 confirmed the fluorescence spectroscopy, MALLS, and QCM findings by providing visual evidence that vaults disassemble into halves as the solution pH is lowered from 6.5 to 3.4.
Vaults are large, hollow ribonucleoprotein particles (13 ( 1 MDa), which are highly conserved among eukaryotic species (1, 2). Vault particles originally were observed in preparations of clathrin-coated vesicles and were named for their resemblance in structure to arched cathedral ceilings (3, 4). The function of vaults in cells is unknown, but they are thought to play a role in nucleocytoplasmic transport on the basis of their association with the nuclear pore complex and nuclear membranes (5, 6). Recent studies also have suggested that vaults may bind to cell signaling pathway components in vivo (7-12). Aside from their native role, externally applied vaults are taken up readily by HeLa cells (13), possibly through endosomal pathways, suggesting that they may be well suited for drug delivery applications. Vaults are composed of multiple copies of an untranslated RNA (vRNA) and three proteins: major vault protein (MVP),1 which makes up 75% of the vault mass; vault poly(ADP-ribose) polymerase (VPARP); and telomerase-associated protein 1 (TEP1) (14). According to cryoelectron microscopy reconstruction images, vaults have a barrel-like shape with maximum dimensions of 72.5 nm × 41 nm × † This work was supported by National Science Foundation NIRT Grant MCB-0210690. * To whom correspondence should be addressed: Chemical and Biomolecular Engineering Department, University of California, 5531 Boelter Hall, 420 Westwood Plaza, Los Angeles, CA 90095. E-mail:
[email protected]. ‡ Chemical and Biomolecular Engineering Department. § These authors contributed equally to this work. | Department of Biological Chemistry. ⊥ California NanoSystems Institute.
41 nm (14) and an internal volume of 5 × 104 nm3 (15). Their capsular structure gives them the potential to be used as compartments in which to encapsulate, transport, and possibly synthesize materials. Each vault is composed of two identical halves, and 96 copies of MVP (48 in each half) form the backbone of the vault structure (14). An illustration of an intact vault, as well as schematics of other vault conformations which will be discussed throughout this work, is presented in Figure 1. When intact vaults are deposited on polylysine-coated mica and imaged by freeze-etch electron microscopy with platinum shadowing, they appear as flowerlike structures with eight rectangular petals (2). These “flowers” are usually seen in pairs, suggesting that each whole vault is composed of two flowers, folded so that the ends of their petals meet along the waist of the vault. It seems likely that vaults in cells open and close in response to cellular signals. However, whether vaults spread fully into flowerlike structures in vivo is unknown. This study was conducted using recombinant CP-MVP vault particles, which consist of 96 copies of a modified MVP and no other proteins or nucleic acids. Recombinant CPMVP subunits can self-assemble into vault particles, which 1 Abbreviations: MVP, major vault protein; VPARP, vault poly(ADP-ribose) polymerase; TEP1, telomerase-associated protein 1; MALLS, multiangle laser light scattering; QCM, quartz crystal microbalance; TEM, transmission electron microscopy; MUA, 11mercaptoundecanoic acid; BCA, bicinchoninic acid; BSA, bovine serum albumin; SAM, self-assembled monolayer; cryoEM, cryoelectron microscopy.
10.1021/bi0606243 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/16/2007
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Goldsmith et al. mented in the study of conformational changes of adsorbed vaults, and transmission electron microscopy (TEM) is employed as an additional means of confirming the interpretation of fluorescence, MALLS, and QCM data. Understanding physiological signals that can trigger vault conformational change in solution will provide insight into native vault behavior, which will aid in the design of vaults as drug delivery vehicles whose contents will be released in response to specific conditions in vivo. Since vaults are known to associate with cell signaling pathway components (7-12), vault conformation may play a key role in cell signaling mechanisms and transport processes. Despite the fact that the details of vault function are unknown, their highly conserved structure across diverse phyla, from worms to humans, strongly suggests an important role that could one day be exploited for therapeutic applications.
FIGURE 1: Schematic illustrating four vault conformations and their corresponding negative stain electron microscopic images. (a) CryoEM reconstruction of an intact vault particle, side view (left), and negative stain EM of a vault particle, side view (right). (b) Two cryoEM orientations of a half-vault derived from splitting a vault at the waist (left). The bottom half-vault is a slightly skewed image which most closely approximates the negative stain EM halfvault image shown at the right. Negative stain EM of a half-vault (right). (c) CryoEM reconstruction of a half-vault pair, top view (left), and negative stain EM of a half-vault pair (right). (d) A model of a half-vault end-on view which has “opened” into an eight-petal flower, based on the open vaults described in Kedersha et al. (2) (left). Negative stain EM image of an open half-vault flower (right) (the flower petals which can be best viewed by freeze-etch platinum shadowing are not visualized as clearly by negative staining).
have been shown to have structures identical to and biochemical characteristics similar to those of endogenous rat vaults (16). In these CP-MVP vaults measuring 73.7 nm × 41 nm × 41 nm (14), each MVP subunit is appended at its N-terminus with a 12-amino acid peptide derived from the metallothionine protein that contains four cysteine residues. Since all MVP N-termini are found at the vault waist (14), it is possible that the cysteine residues of CPMVP form inter- and/or intrachain disulfide bridges that stabilize the vault structure (14). CP-MVP vaults in fact are found to be more stable than wild-type particles, with minimal variations in size, shape, and conformation (14); however, there is no direct evidence to implicate a role for disulfide bridges in improved vault stability. Although CPMVP vault samples purified using sucrose gradients (14, 16) are the most stable of all recombinant and native vault types prepared to date, small amounts of aggregated and denatured particles are still visible in CP-MVP preparations (as discussed in Results and Discussion). Inspired by previous findings of vault dissociation on polylysine-coated surfaces (2), our work is the first to employ several experimental tools in the study of vault conformational change in solution in response to low pH. Preliminary ideas for possible means of effecting conformational change in vaults, including exposure to low pH, were obtained from prior studies of structural change induced in viruses by manipulation of their solution environments (17-20). This study builds on the prior results of others showing the usefulness of fluorescence spectroscopy (21-23), fluorescence quenching (24-27), and multiangle laser light scattering (MALLS) (28-34) for the monitoring of conformational changes of proteins and virus particles. Here, the quartz crystal microbalance (QCM) (35-38) also has been imple-
MATERIALS AND METHODS CP-MVP recombinant vaults were prepared as described previously (14, 16) and were stored at 4 °C in 20 mM MES at pH 6.5 until they were used. In short, the cDNA sequence of MVP was subcloned into a recombinant baculovirus, where it was overexpressed, and then transfected into an Sf9 insect cell line (14). Recombinant vaults were extracted from lysed insect cells and purified using centrifugation and a sucrose gradient. They are found mostly in the 40 and 45% sucrose layers, which coincides with natural vaults purified from tissues (14, 16). To change the pH environment of the vaults, vault stock aliquots were diluted at least 15-fold into the desired buffer and were allowed to equilibrate for 1 h. Materials. MES, Tris, L-tryptophan, and 11-mercaptoundecanoic acid (MUA) were obtained from Sigma Chemical Co. (St. Louis, MO). The pH of MES (20 mM, pH 6.5) and 85 mM Tris buffers, prepared in the pH range of 6.5-9.0, was adjusted using 4 M HCl and 4 M NaOH. Citratephosphate buffers in the pH range of 2.6-6.5 (total concentration of 55-85 mM) were prepared using anhydrous citric acid and sodium phosphate dibasic heptahydrate (Fisher Chemicals, Fair Lawn, NJ). Acrylamide was purchased from Invitrogen (Carlsbad, CA). Fluorescence Spectroscopy. Intrinsic aromatic amino acid (principally tryptophan) emission spectra (FL3-22, Instruments S.A., Inc., SPEX Division, Edison, NJ) were gathered at a controlled pH at an excitation wavelength of 295 nm and were background-corrected. In studies of intrinsic fluorescence quenching by acrylamide, aliquots of a 2 M acrylamide stock were added to the pH-buffered vault solution, which was allowed to equilibrate for 10 min before spectra were recorded as described above. Spectra were corrected for the inner filter effect due to acrylamide adsorption at the excitation and appropriate emission wavelengths (39). For proteins, emission is heterogeneous and the appropriate equation for the fraction of tryptophan (Trp) fluorescence quenched (40) is
F0 - F F0
fiKQi[Q] )∑ i 1 + KQi[Q]
(1)
where F0 is the peak fluorescence emission intensity of vaults in the absence of quencher, F is the fluorescence intensity
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in the presence of acrylamide at a molar concentration ([Q]), fi is the fractional contribution of component i to the total fluorescence, and KQi is the collisional quenching constant (inverse molar), also called the Stern-Volmer constant, of component i. For proteins containing multiple Trp residues (for example, each CP-MVP vault particle contains 576), eq 1 can be expanded for the i residues. Since fitting the expanded form of eq 1 is not practical for vaults, we make the approximation that at low quencher concentrations, [Q]2 and higher-order terms may be eliminated. Therefore, peak fluorescence intensity data at low acrylamide concentrations (
